Coronary heart disease is the leading cause of death in the U.S., and the leading cause of death associated with smoking. Smoking poses a significant, detrimental impact on the heart, and the toxins in cigarette smoke cause plaques to form in the arteries, which oftentimes leads to atherosclerosis. Atherosclerosis is just one of several types of arteriosclerosis, which is characterized by thickening and hardening of artery walls (e.g., coronary arteries, carotid arteries, aorta, ileofemoral arteries). Over time, this material thickens, hardens and may eventually block or severely narrow the arteries. More than 61 million Americans suffer from some form of cardiovascular disease, including high blood pressure, coronary heart disease, stroke, congestive heart failure, and other conditions. More than 2,600 Americans die every day because of cardiovascular diseases; about 1 death every 33 seconds.
ApoA-I, a major component of high density lipoprotein (“HDL”), has been shown to have anti-atherogenic properties. Apolipoprotein A-IMilano (“ApoA-IMilano”) is a mutant form of ApoA-I with a single amino acid substitution (Arg173 to Cys173) (Weisgraber, K. H. et al., “A-IMilano apoprotein, isolation and characterization of a cysteine-containing variant of the A-I apoprotein from human high density lipoproteins,” J Clin Invest, Vol. 66, pp. 901-7 (1980)). This mutation appears to confer greater resistance to atherosclerosis in individuals with this genotype (Franceschini, G. et al., “A-IMilano apoprotein. Decreased high density lipoprotein cholesterol levels with significant lipoprotein modifications and without clinical atherosclerosis in an Italian family,” J Clin Invest, Vol. 66, pp. 892-900 (1980)). The structural alteration of ApoA-Imilano is associated with a higher kinetic affinity for lipids and an easier dissociation from lipid protein complexes, which contributes to its accelerated catabolism and increased uptake of tissue lipids (Franceschini, G. et al., “Apolipoprotein A-IMilano. Accelerated binding and dissociation from lipids of a human apolipoprotein variant,” J Biol Chem, Vol. 260, pp. 16321-5 (1985)). ApoA-I serves an anti-atherogenic role by functioning to reverse cholesterol transport, reduce oxidized lipids within HDL, prevent foam cell formation, inhibit platelet activation, as well as having anti-inflammatory, anti-oxidant and anti-thrombotic effects. However, the exact mechanism by which ApoA-IMilano confers its resistance to atherosclerosis remains to be elucidated.
It has previously been reported that 5 intravenous injections of recombinant apo A-I milano reduces high cholesterol and balloon injury induced ileofemoral atherosclerosis in rabbits (Ameli, S. et al., “Recombinant apolipoprotein A-I Milano reduces intimal thickening after balloon injury in hypercholesterolemic rabbits,” Circulation, Vol. 90, pp. 1930-1941 (1994); in addition repeated intravenous injections of a 40-80 mg/kg dose of recombinant ApoA-IMilano (rApoA-IMilano) over a 5-week period induces a significant reduction/regression in aortic atherosclerosis in ApoE-deficient mice compared with untreated controls (Shah, P. K. et al., “Effects of recombinant apolipoprotein A-I(Milano) on aortic atherosclerosis in apolipoprotein E-deficient mice,” Circulation, Vol. 97, pp. 780-5 (1998)). Furthermore, a single large intravenous dose of recombinant Apo A-I milano was shown to reduce lipid content and inflammation in aortic sinus plaques of ApoE knockout mice within 48 hours leading to a more stable plaque phenotype (Shah, P. K. et al., “High-Dose Recombinant Apolipoprotein A-IMilano Mobilizes Tissue Cholesterol and Rapidly Reduces Plaque Lipid and Macrophage Content in Apolipoprotein I-Deficient Mice: Potential Implications for Acute Plaque Stabilization,” Circulation, Vol. 103, pp. 3047-3050 (2001). Additional studies using rApo A-I milano have also demonstrated its ability to improve endothelial dysfunction in ApoE knockout mice and reduce in stent stenosis in a porcine model when applied at the site of stent implantation (Kaul, S. et al., “Intramural Delivery of Recombinant Apolipoprotein A-IMilano/Phospholipid Complex (ETC-216) Inhibits In-Stent Stenosis in Porcine Coronary Arteries,” Circulation, Vol. 107, pp. 2551-2554 (2003); Kaul, S. et al., “Rapid reversal of endothelial dysfunction in hypercholesterolemic apolipoprotein E-null mice by recombinant apolipoprotein A-I-phospholipid complex,” J Am Coll of Cardiology, Vol. 44, Issue 6, pp. 1311-1319 (2004). In a more recent double-blind, randomized, placebo-controlled study, it was demonstrated that infusion of recombinant ApoA-IMilano-phospholipid complexes in patients with acute coronary syndromes with five doses at weekly intervals produced significant regression of the coronary atheroma burden (Nissen, S. E. et al., “Effect of recombinant ApoA-I Milano on coronary atherosclerosis in patients with acute coronary syndromes: a randomized controlled trial,” JAMA, Vol. 290, pp. 2292-300 (2003)). These findings suggest that therapy based on ApoA-IMilano could have potential as an effective anti-atherogenic strategy. However, frequent intravenous injections and expenses related to the production of recombinant proteins limit the practical and widespread applicability of this approach in humans.
Gene delivery is a promising method for the treatment of acquired and inherited diseases. A number of viral-based systems for gene transfer purposes have been described, such as retroviral systems, which are currently the most widely used viral vector systems for gene transfer. For descriptions of various retroviral systems, see, e.g., U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109. However, the recent description of retrovirus vector-associated leukemogenesis in two patients has underscored potential limitations of this vector system.
A number of adenovirus-based gene delivery systems have also been developed. Human adenoviruses are double-stranded DNA viruses which enter cells by receptor-mediated endocytosis. These viruses are particularly well suited for gene transfer because they are easy to grow and manipulate and they exhibit a broad host range both in vivo and in vitro. Adenovirus is easily produced at high titers and is stable so that it can be purified and stored. For descriptions of various adenovirus-based gene delivery systems, see, e.g., Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921; Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al. (1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993) Human Gene Therapy 4:461-476. However adenovirus virus vectors, including the newer helper-dependant adenovirus vectors are associated with triggering host innate immunity that can be highly toxic.
In an earlier study that utilized a recombinant human ApoA-I adenovirus, De Geest et al. demonstrated transient expression of wild-type human ApoA-I in ApoE-deficient mice of over 150 mg/dl peaking at 6 days (De Geest, B. et al., “Effects of adenovirus-mediated human apo A-I gene transfer on neointima formation after endothelial denudation in apo E-deficient mice,” Circulation, Vol. 96, pp. 4349-56 (1997)). In a similar study, tsukamoto et al. showed that intravenous injection into ApoE-deficient, low density lipoprotein receptor-deficient and wild-type C57BL/6 mice resulted in mean peak plasma human ApoA-I concentrations of 235, 324 and 276 mg/dl, respectively, after 3 days post-injection and declined thereafter (Tsukamoto, K. et al., “Comparison of human apoA-I expression in mouse models of atherosclerosis after gene transfer using a second generation adenovirus,” J Lipid Res, Vol. 38. pp. 1869-76 (1997)). The overall decrease in levels of human ApoA-I transgene expression may be attributed to an inflammatory response to virally infected cells (Engelhardt, J. F. et al., “Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver,” Proc Natl Acad Sci USA, Vol. 91, pp. 6196-200 (1994)). In another study, gene transfer with an adenovirus and the 256-bp ApoA-I promoter, the genomic ApoA-I DNA, and four ApoE enhancers resulted in ApoA-I expression above 20 mg/dl for up to 6 months (De Geest, B. et al., “Sustained expression of human apolipoprotein A-I after adenoviral gene transfer in C57BL/6 mice: role of apolipoprotein A-I promoter, apolipoprotein A-I introns, and human apolipoprotein E enhancer,” Hum Gene Ther, Vol. 11, pp. 101-12 (2000)). However, there is no data with regards to the effective concentration of circulating ApoA-IMilano. Rather, past studies have suggested that the circulating levels of serum proteins do not necessarily correlate with their effective biological concentration. For example, bone marrow transplantation studies using ApoE+/+ donor and ApoE−/− recipient mice showed significant improvements in atherosclerotic lesions in recipient mice with 10% chimerism (Sakai, Y. et al., “Bone marrow chimerism prevents atherosclerosis in arterial walls of mice deficient in apolipoprotein E,” Atherosclerosis, Vol. 161, pp. 27-34 (2002)). Other investigators have also examined the effect of macrophage-derived ApoE by bone marrow transplantation with wild-type marrow (Linton, M. F. et al., “Prevention of atherosclerosis in apolipoprotein E-deficient mice by bone marrow transplantation,” Science, Vol. 267, pp. 1034-7 (1995); Boisvert, W. A. et al., “Treatment of severe hyper-cholesterolemia in apolipoprotein E-deficient mice by bone marrow transplantation,” J Clin Invest, Vol. 96, pp. 1118-24 (1995); Van Eck, M. et al., “Bone marrow transplantation in apolipoprotein E-deficient mice. Effect of ApoE gene dosage on serum lipid concentrations, (beta)VLDL catabolism, and atherosclerosis,” Arterioscler Thromb Vasc Biol, Vol. 17, pp. 3117-26 (1997)). These studies demonstrated normalization of plasma cholesterol 4-5 weeks post-transplant and reduction of atherosclerosis 14-20 weeks post-transplant. The level of circulating ApoE varied from 3.8% to 12.5% of normal levels in C57BL/6 mice (Linton, M. F. et al. (1995); Van Eck, M. et al. (1997); Spangenberg, J. et al., “Influence of macrophage-derived apolipoprotein E on plasma lipoprotein distribution of apolipoprotein A-I in apolipoprotein E-deficient mice,” Biochim Biophys Acta, Vol. 1349, pp. 109-21 (1997)). Therefore, considerably lower levels of the circulating bioactive serum molecules may be sufficient for an effective biological action on the vessel wall.
In a recent study, Van Linthout et al. used a “gutted” helper-virus independent adenoviral vector and an expression cassette consisting of the human αl-antitrypsin promoter, the human genomic ApoA-I DNA and four copies of the human ApoE enhancer in an effort to improve duration of ApoA-I transgene expression (Van Linthout, S. et al., “Persistent hepatic expression of human apo A-I after transfer with a helper-virus independent adenoviral vector,” Gene Ther, Vol. 9, pp. 1520-8 (2002)). The investigators were able to produce long-term and high levels of ApoA-I expression (170±16 mg/dl at 6 months) in C57BL/6 mice. However, there are still some concerns of toxicity in using these adenoviral vectors in humans (St George, J. A., “Gene therapy progress and prospects: adenoviral vectors,” Gene Ther, Vol. 10, pp. 1135-41 (2003)). In the case of AAV vectors, the parental wild-type AAV is non-pathogenic (Muzyczka, N., “Use of adeno-associated virus as a general transduction vector for mammalian cells,” Curr Top Microbiol Immunol, Vol. 158, pp. 97-129 (1992)).
The construction of recombinant adeno-associated virus (“rAAV”) vectors has been described. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Patent Publication Numbers WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (published Mar. 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Gao, G. (2002) Proc Natl Acad Sci USA 99:11854-11859; Hauck, B. (2003) Journal of Virology 77(4):2768-2774; Gao, G. (2004) Journal of Virology 78(12):6381-6388.
Eukaryotic vectors based upon the nonpathogenic parvovirus, adeno-associated virus (“AAV”), have recently emerged as promising vehicles for efficient gene transfer. AAV is a replication-defective DNA virus with a 4.7 kb genome with palindromic inverted terminal repeats (“ITR”). Coinfection with a helper virus, typically adenovirus or herpes simplex virus, is required for productive infection. In the absence of helper virus coinfection, AAV stably integrates via the ITRs into chromosomal DNA, or may persist in an episomal state. Wild type AAV is unique in the capacity for integration into a specific region of human DNA termed “AAVS1” on human chromosome 19. AAV have been found in many animal species, including nonhuman primates, canines, fowl, and humans (Murphy, F. A. et al., “Classification and nomenclature of viruses: sixth report of the International Committee on Taxonomy of Viruses,” Arch. Virol., Vol. 1995, pp. 169-175 (1995)). There are more than 100 serotypes of AAV, including AAV type 1 (AAV-1), isolated from primates, AAV-2, AAV-3, and AAV-5, isolated from humans, and AAV-6, isolated from a human adenovirus preparation; other serotypes are being intensively evaluated for use in gene therapy. See, e.g. Gao, P. (2004) J. Virol.; 78(12):6381-6388. AAV-2 is the most characterized primate serotype, since its infectious clone was the first one made (Samulski, R. J. et al., “Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells,” Proc. Natl. Acad. Sci. USA, Vol. 79, pp. 2077-2081 (1982)). The full sequences for AAV-3A, AAV-3B, AAV-4, and AAV-6 recently were determined (Chiorini, J. A. et al., “Cloning of adeno-associated virus type 4 (AAV4) and generation of recombinant AAV4 particles,” J. Virol., Vol. 71, pp. 6823-6833 (1997); Muramatsu, S. et al., “Nucleotide sequencing and generation of an infectious clone of adeno-associated virus 3,” Virology, Vol. 221, pp. 208-217 (1996); Rutledge, E. A. et al., “Infectious clones and vectors derived from adeno-associated virus (AAV) serotypes other than AAV type 2,” J. Virol., Vol. 72, pp. 309-319 (1998)). Generally, all primate AAV show more than 80% homology in nucleotide sequence. AAV vectors have been based primarily on serotype 2, a human-derived parvovirus (Parks, W. P. et al., “Seroepidemiological and ecological studies of the adenovirus-associated satellite viruses,” J. Virol., Vol. 2, pp. 716-722 (1970); Samulski, R. J., “Adeno-associated virus: integration at a specific chromosomal locus,” Curr. Opin. Genet. Dev., Vol. 3, pp. 74-80 (1993)). The early availability of an infectious clone of AAV-2 stimulated work on the development of replication-defective vectors. The AAV2 genome has two major open reading frames (“ORFs”); the left encodes functions necessary for AAV ori mediated replication and site specific integration (Rep), while the right encodes functions necessary for encapsidation (Cap). AAV vectors transduce many different types of cells. Multiple studies have amply demonstrated that rAAV vectors can transduce quiescent, nonproliferating targets. rAAV vectors do not encode any viral encoded genes, reducing their intrinsic immunogenicity. In addition, prolonged in vivo transgene expression following rAAV transduction has been documented in animal models. Finally, since its discovery in the mid-1960s, wild type AAV has yet to be definitively identified as a pathogen in either animals or humans. On the contrary, there is evidence that infection with wild type AAV inhibits transformation by bovine and human papillomaviruses and the activated H-ras oncogene in vitro, and induces apoptosis in p53 deficient, malignant cells, while epidemiologic studies suggest that prior infection in humans may actually confer an oncoprotective effect. Thus, for reasons outlined above and supported by data described herein, AAV-based vectors are well suited for the stable introduction of transgenes into hematopoietic cells.
Thus, there is a need in the art for a gene therapy strategy for the treatment of atherosclerosis and the array of diseases and physiological conditions related to the same. It is likely that the use of rAAV vectors would be virtually harmless for in vivo gene transfer, and may offer a viable approach for elevated and sustained levels of ApoA-IMilano expression. Therefore, a strategy for gene delivery and expression, which would result in constitutive and persistent levels of circulating ApoA-I and/or ApoA-IMilano protein, particularly when delivered via rAAV vector technology, is especially desirable.